The Regulation Of E3 Ligase SLL8 On Pollen Development In Tomato

The development of flowers plays a very important role in the reproduction of the offspring and the quality of fruit in tomato.The pollen germination and the elongation of pollen tube are the precondition for the successful double fertilization of tomato.Besides,the pollen germination plays an important role in the process of tomato fertilization.Therefore,exploring the development of flower plays an important role in the growth and development of tomato.Ubiquitin protein degradation pathway is a major protein degradation pathway widely existed in plants.E3 ubiquitin ligases are one of the key enzymes in this process.For plants,the researches on E3 protein are mainly in Arabidopsis thaliana including flowering time,self-incompatibility and so on.However,our understanding of the regulation of E3 ligases on pollen development in plants is scanty,especially in horticultural plants.The tomato(Solanum lycopersicum)plants were used as experimental materials,selecting high expression of E3 ubiquitin ligase SLL8 in stamen.In addition,we identified that SLL8 had E3 ubiquitin ligase activity ubiquitination assay in vitro.We also constructed SLL8 gene deletion mutant by CRISPR/Cas9 and SLL8 overexpressing transgenic plants.Finally,the functions of SLL8 gene using mutants and overexpressing plants were studied in pollen development.The main results were as follows:1.Screening of E3 ubiquitin ligase SLL8.We identified 18 homologous sequences of E3 ubiquitin ligases genes in tomato,the homologous of E3 ligases genes in Arabidopsis thaliana,through the amino acid sequence alignment.18 tomato E3 ubiquitin ligase genes were analyzed for q RT-PCR.As a result,three genes in flower were significantly higher than those in leaf,and three genes were named SLL8,BRG3 and SHE4 respectively.Tomato SLL8,BRG3 and SHE4 were highly expressed in stamen,while the relative expression of SLL8 was significantly higher in the mature pollen stage of microspore development than the rest of microspore development.Besides,the relative expression of SLL8 was significantly up-regulated under heat stress.The results showed that SLL8 was highly expressed in stamen especially in the mature pollen stage of microspore development and was involved under heat stress.2.Analysis of E3 ubiquitin ligase activity for SLL8 and the acquisition of genetically modified materials.According to the comparison of the AT1G76410 protein sequence in Arabidopsis thaliana,we found that the homologous protein SLL8 in tomato.SLL8 had a highly conserved domain RING-H2,which contained only one exon and no introns.The similarity and identity were 63.96% and 55.03%respectively.The MBP-SLL8 fusion protein was obtained in prokaryotes and ubiquitination assay in vitro was carried out.The experimental results showed that SLL8 was an E3 ubiquitin ligase,which had the activity of E3 ubiquitin ligases.SLL8 protein existed in the nucleus and cytoplasm in tobacco.Two overexpressing lines of SLL8,SLL8OE-1# and SLL8OE-2#,were obtained.There were no differences in seedling growth and flower morphology between mutant sll8 plants,WT and overexpressed plants.However,the seed germination rate in the mutant sll8-2 and sll8-4 was 60% and 65%,respectively,which were significantly lower than those of WT and overexpressed lines of SLL8OE-1# and SLL8OE-2# plants.In conclusion,SLL8 had the activity of ubiquitin ligase and was expressed in both cytoplasm and nucleus,which affected the seed germination in tomato.3.The roles of SLL8 in the pollen development.The results showed that the pollen viability rate of SLL8OE1# and SLL8OE2# plants was significantly higher than that of WT plants,which increased by 11.11% and 18.06% respectively,while the pollen germination rate showed the same trend.However,the pollen viability rate of the mutant sll8-1 and sll8-4 was significantly lower than that of the control,which decreased by 44.44% and 36.11% respectively.In addition,the pollen germination rate also showed the same trend,which was significantly lower than that of the WT plants.Through SEM observation,some malformations and adhesions appeared in the pollen of sll8-1 and sll8-4.However,there was no significant difference in the development of the tapetum with the WT plants.SLL8 was up-regulated in different heat-resistant tomato varieties under heat stress.In addition,the pollen viability and germination rate of SLL8OE1# and SLL8OE2# plants under heat stress were significantly higher than those of WT plants,while mutant sll8-1 and sll8-4 were significantly lower than those of WT plants.In summary,SLL8 affected pollenviability and pollen germination and caused abnormal pollen,but it did not participate in the development of the tapetum.